ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library Cryptococcus Spp

CONTRIBUTION OF PATHOLOGY TO THE DIAGNOSIS OF FUNGAL INFECTIONS

DR GEETIKA KHANNA PROFESSOR OF PATHOLOGY © by CIOauthorLABORATORY VMMC & SAFDARJUNG HOSPITAL NEW DELHI ESCMID Online Lecture Library PATHOLOGY – RELEVANCE IN FUNGAL DISEASE

1. Rapid & cost-effective means of arriving at a presumptive diagnosis …Culture may take wks …Organism may not grow at all.

2. Allows definitive interpretation© by inauthor cases with specific findings - unique fruiting head of Aspergillus spp. - atESCMID least 1 unequivocal, Online intact, endospore Lecture-filled spherule Library of Coccidioides . 3. Provides insight into the diagnostic significance of culture isolates ….gives information on vascular/tissue invasion & host reaction ….address the frequently encountered problems of contamination & infection vs colonization.

4. May be the only method of diagnosis for fungal pathogens like Pneumocystis & Lacazia loboi. © by author 5. SometimesESCMID detects Online fungal disease Lecture in clinically Library unsuspected cases in which no culture has been sent. Overview of special stains for fungi HPE: 1. PAS [red] & GMS [black]: • Both highlight the cell wall - Used as screening tools. • GMS more sensitive than PAS…signal to noise issue..stains lysosomes, inflammatory cells & tissue reticulin. • PAS gives a better visualization of surrounding tissue compared to GMS. • GMS with H&E counterstain - best combination for fungus & host reaction.

3. Mucicarmine & Alcian Blue [red & blue]: Capsule of Cryptococcus © by author 4. Fontana-Masson [black] : Cryptococcus & dematiaceous fungi & some speciesESCMID of Aspergillus, MucoralesOnline & Trichosporon. Lecture Library Cytology (sputum, BAL, CSF, FNA): Calcoﬂuor white, Uvitex, MGG, Pap, PAS, GMS. Reporting of fungal infections in tissue & cytological preparations 1. Describe the fungal elements.  Yeast / hyphae / Both/ endosporulating structures.  Yeast forms – Diameter, Budding or Fission/Type of budding (narrow based or broad based).  Hyphae - Width, septations, branching angle, dilated bizarre forms (Mucorales), Brown pigmentation (Dematiaceous fungi).  Also comment on quantity of fungal elements & viability.

2. Clearly state the fungus which is most frequently associated with the described morphology & enlist the fungi that can display a similar morphology. © by author 3. Identify the inﬂammatory reaction, comment on presence of ESCMIDvascular or tissue Online invasion, necrosis, Lecture or hemorrhage. Library 4. Correlate all pathological findings with clinical features, epidemiology & results of alternative testing if available . Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80. MORPHOLOGICAL CLASSIFICATION OF FUNGAL DISEASES  Diseases Where Yeasts or Yeast-Like Structures Are Usually Seen  Diseases Where Hyphae Are Usually Seen  Diseases caused by Fusarium, Scedosporium, and other hyaline septated molds  Diseases caused by Bipolaris/Curvularia and other Dematiaceous fungi  Dermatophyte disease  Diseases caused by other© bymolds author (coelomycetes)

*TheESCMID “clinical spectrum Online & host tissue Lecture response “Library to every fungus is as important as the “morphology of the fungus” & must be kept in mind while making a diagnosis. DISEASES WHERE YEASTS OR YEAST-LIKE STRUCTURES ARE USUALLY SEEN IN TISSUES

 Blastomycosis  Cryptococcosis  Histoplasmosis  Coccidioidomycosis  Candidiasis© by author ESCMID Online Lecture Library Blastomyces dermatitidis Morphology of fungus Clinical spectrum & host tissue D/Ds response •Yeasts meas. 8 -15 μm. • Acute pneumonia: Mixed 1. Larger forms to be suppurative response. differentiated from • Show single, broad-based • Chronic pneumonia: a) Immature spherules budding. Pyogranulomatous inflammation of C. immitis. with numerous multinucleate cells. b) Yeast forms of • Clear space around the • Cutaneous/mucosal lesions: P. Brasiliensis. fungal cell on H&E staining - Marked epithelial hyperplasia. c) Conidia of (corresponds to a thick - Neutrophilic microabscesses in aspergillus. refractile cell wall); stains epithelium. with silver stains. - Chronic inflammation in the dermis/ 2. “Microforms” to submucosal© by tissue. author be differentiated • Larger forms may • Disseminated lesions: Various from the occasionally be accompanied inflammatory responses depending Yeast forms of by the “microESCMID forms”. Onlineon immune status. Lecture LibraryCandida spp., *Tuberculosis or neoplasia can be Histoplasma, present concomitantly Cryptococcus. © by author ESCMID Online Lecture Library

Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80. PAS stain.

Morphology of fungus Clinical spectrum & host tissue D/Ds response • Spherical-oval yeasts. • Pneumonia & Cryptococcomas: Candida & • Marked variation in size. Granulomatous reaction with fibrosis. Histoplasma - • Narrow-based budding. Occasionally pseudotumour formation. similar in size & can • Thick polysaccharide • Neutrophils unusual – indicate be confused capsule. bacterial superinfection/response to histologically. • Pseudohyphae (with massive necrosis. massive yeast proliferation). • Disseminated disease: Varying infl. Well encapsulated response depending on immune status. organisms easily India ink: Negative stain - • Immunocompetent individuals: Well differentiated using highlights the capsule. formed granulomas with few organisms. capsular stains. Mucicarmine & Alcian blue: • Severe© by T cell author deficiency: Capsule. Minimal inflammation with abundant Organisms with PAS & GMS: Cell wall. intra/extracellular organisms (sheets of poorly formed MasonsESCMID-Fontana: Yeast cell Onlineyeast filled lacunae Lecture- “soap bubble Librarycapsule pose some (permits differentiation from lesion”). problem and need most other pathogenic yeasts to be differentiated except Trichosporon beigelii). by melanin stains. CDC public health image library

- Khanna G, Bhattacharya SN, Singhal A,© Singh by N, Sharma author S. Fatal disseminated cryptococcosis in a HIV negative elderly Indian woman - early diagnosis by polarizing microscopy. Indian J Pathol Microbiol 2004; 47(4): 542-44. - Randhawa HS, Chowdhari A, Khanna G. Comment on Singh et al’s “Cryptococcosis in a bandicoot rat.” Med MycolESCMID2007; 45:7, 655-6 Online Lecture Library - Capoor MR, Khanna G, Malhotra R, Verma S, Nair D, Deb M, Aggarwal P. Disseminated cryptococcosis with necrotizing fasciitis in an apparently immunocompetent host: a case report. Medical Mycology 06/2008; 46(3):269-73. PAS stain.

© by author ESCMID Online Lecture Library Histoplasma Morphology of fungus Clinical spectrum & host tissue response D/Ds H. Capsulatum: • Spectrum varies from localized granuloma Include all small • Oval 2-4-μm yeast formation to massive proliferation of MPs sized yeasts which show containing yeast cells. • Narrow-based budding. narrow based • Acute pneumonia: Intra-alveolar aggregates budding & • H&E: Halo corresponding of histiocytes, expand to form nodules of Intracellular to the cell wall. parenchymal/ vascular necrosis. clustering. • Chronic histoplasmosis: Multiple small • GMS & PAS: Highlight the granulomata seen all over, commonly in pleura cell wall. & spleen…May get hyalinized...Rarely residual • Intracellular clustering necrosis© with by yeast author forms may be seen. within macrophages - • Mediastinitis: Massive hilar & mediastinal diagnostic hallmark. LAP; lamellar fibrosis & calcification (fibrosing ESCMID Onlinemediastinitis); organismsLecture rarely found/if Library found • In African histoplasmosis the yeast swollen, distorted, no budding..? non-viable). size is larger (8-15μm) & yeast may be pigmented. Fungus to be Differentiating features differentiated

“Micro” forms of B. Presence of larger forms & broad based budding dermatitidis Capsule deficient Show marked size variation & positive melanin staining Cryptococci Endospores of Presence of remnants of a ruptured spherule or an intact spherule. Coccidioides spp. Pneumocystis jirovecii Does not show budding & has a prominent intracystic focus.

Penicillium marneffei Fission,© sausage by shape,author transverse septum. Candida glabrata Typically extracellular; no halo, shows greater size variability & pure neutrophilic infiltrate. ESCMID Online Lecture Library Leishmaniasis & H&E stains entire organism; no halo; in leishmaniasis kinetoplast toxoplasmosis seen by the side of the nucleus; in toxoplasmosis infected cells are cardiomyocytes & neurons rather than histiocytes. © by author ESCMID Online Lecture Library

CDC public health image library PAS stain

PAS stain

GMS stain

© by author ESCMID Online Lecture Library Coccidioides immitis Morphology of fungus Clinical spectrum & host tissue D/Ds response • Thick walled spherules • Pulmonary & cutaneous lesions R. seeberi: (10-100 μm) which demonstrate a mixed suppurative- • Palatal & nasopharyngeal contain multiple granulomatous response with polyps. endospores (2-5 μm). abundant eosinophils. • Larger sporangia & • Rupture to release the • Eosinophils secrete EBP which endospores. endospores into the induces an intense rim of • 50-100mm, can be seen surrounding tissues. eosinophilic material around fungal with the naked eye as yellow • H&E stains both the elements. “Splendore-Hoeppli pin-head sized dots in a spherules & endospores. phenomenon”. polyp. • GMS & PAS stain the • Immunosuppressed - marked spherule wall & necrosis© without by granulomas author Endospores/Young endospores. formation. spherules without • PAS staining fades with • Occ. mycelia may be seen in the endospores can be confused the maturityESCMID of the cavitary Online or skin lesions. Lecture withLibraryBlastomyces, organism. Emmonsia, Candida & Histoplasma. *Pneumocytis & Coccidioides can co-exist. PAS stain

CDC public health image library collection H&E stain

Capoor MR, Khanna G, Rajni, Batra K, Nair D, Venkatchalam VP, Aggarwal P. Rhinosporidiosis in Delhi, North India: Case Series from a Non-endemic area and Mini-review. Mycopathologica 2009 Apr 5. 168(2):89-94. Candida spp. Morphology of fungus Clinical spectrum & host tissue D/Ds response • C. albicans: Mats of •.Superficial & invasive disease. • C. albicans - to be yeasts measuring 3-5 μm • HPE very important to define differentiated from Aspergillus in dm intermingled with invasion. spp. (presence of true pseudohyphae • Neutrophilic inflammation with septations). & Trichosporon (filaments), which show some lymphocytes and spp. (presence of a periodic constrictions but macrophages, fibrin, and constriction between the base not true septations. coagulative necrosis. Rarely few of the blastospore & the germ giant cells and granulomas. tube). • C. glabrata: Does not • Invasion of BVs may cause produce pseudohyphae. mycotic aneurysms & • C. glabrata - to be thrombophlebitis.© by author differentiated from similar sized • Necrotizing vasculitis is seen yeast forms like Histoplasma. but no demonstrable organism ESCMID indicatingOnline that Candida Lecturesoluble Library fraction causes the necrotizing lesions. PAS stain

GMS stain

© by author ESCMID Online Lecture Library DISEASES CAUSED BY OTHER FUNGI & ORGANISMS RESEMBLING FUNGI THAT DISPLAY YEASTS OR YEAST-LIKE STRUCTURES IN TISSUES • Pneumocystis • Sporothrix • Penicillium • Paracoccidioides brasiliensis • R. seeberi. • Emmonsia© by author crescens

- Capoor MR, Ramesh V, Khanna G, Singh A, Agarwal P. Sporotrichosis in Delhi among the migrant population from Uttarakhand, India.Tropical Doctor 01/2011; 41(1):46-8. - CapoorESCMIDM, Nair D, Deb M, Ramesh OnlineV, Khanna G, LectureRajni, Aggarwal P, Chand LibraryR, Chowdhary A, Mussa AY, Randhawa HS. Endemic occurrence of sporotrichosis in Uttaranchal, India – report of two autochthonous cases. Indian J Dermatology 2007; 50(4 suppl11): S10-S13. Fungus Morphology & staining Pathological D/Ds Pneumocystis Cysts 2-6μm in size with an intracystic Small yeasts with narrow based jirovecii thickening called “capsular dot”. budding & intracellular clustering. Stained with GMS, Gram Weigert & Giemsa Penicillium • Dimorphic fungus –Yeast 2-6µm in size. • Small yeasts with narrow based marneffei • Divides by fission (no budding); resemble a budding & intracellular clustering. sausage with transverse septum. • Most striking clinico-pathological similarity with histoplasmosis. Sporothrix • Round-oval-cigar shaped yeast measuring 2- 1. Histoplasmosis (Intracellular schenckii 6µm clustering) • Narrow based or tube like budding. 2. Candida (pseudohyphae) • Can be intra & extracellular. 3. Leishmania (Kinetoplast) • Splendor-Hoeppli phenomenon (40-92% cases). Paracoccidioides • Spherical yeasts; double contoured wall; Yeast forms showing 1-3 buds with brasiliensis size varying between 4-60µm. absence the “pilot wheel • Shows budding with© multiple by tear author drop buds appearance” need to be surrounding the parent cell (pilot wheel differentiated from C.neoformans, appearance) better seen with GMS. S. schenckii & Lacazia loboi. Emmonsia • Dimorphic fungus, inhaled or inoculated in Adiaspores need to be crescensESCMIDthe skin. Online Lecturedifferentiated Library from Coccidioides • Called “Adiaspiromycosis or Haplomycosis” spherules which contain due to presence of adiaspores which are large, endospores while adiaspores are double walled, empty structures measuring empty. 20-400μm. GMS stains the wall to demonstrate fenestrations. © by author ESCMID Online Lecture Library

CDC public health image library collection PAS stain

GMS stain

H&E

1. Small yeasts intermingled with pseudohyphae & hyphae (morphology consistent with Candida; D/D Aspergillosis, Hyaline septated hyphae). 2. Non-pigmented (hyaline) septated hyphae with acute angled branching (morphology consistent with Aspergillosis; D/D Hyaline septated hyphae).. 3. Non-pigmented (hyaline) pauciseptate ribbon like hyphae with right angled branching© by(morphology author consistent with Mucorales; however, Aspergillus spp. & Hyaline septated hyphae canESCMID sometimes be confused).Online Lecture Library 4. Pigmented irregular hyphae & yeast like structures both with septations (morphology consistent with Dematiaceous fungi). Aspergillus spp. Morphology & Clinical spectrum & host tissue response D/Ds staining • Thin hyphae (3-12 1. Allergic aspergillosis(ABPA/AFRS): 1. Other Hyaline μm). AM with non-invasive hyphae; bronchial wall septated molds • Septate. may show E, N, MPs, granulomas, vasculitis & (Hyalino- • Show acute-angle interstitial ﬁbrosis. hyphomycetes): (45o) or 2. Chronic colonizing aspergillosis/ 1. Fusarium spp. dichotomous aspergilloma: Two types- 2. Scedosporium branching. a)Thin walled aspergillomas (fungal ball spp. • Vesicles with surrounded by fibrosis). 3. Trichoderma spp. conidia (when the b)Chronic cavitary/necrotizing aspergilloma (a 4. Paecilomyces fungi are present in necrotic layer with abundant hyphae surrounded spp. cavitary lesions or by granulation© tissueby & authoran outer layer of skin). fibrosis). 2. Sometimes 3. Invasive aspergillosis: mucorales. • A. NigerESCMIDinfection Angioinvasion Onlinemay lead Lecture to hemorrhage, Library usually shows calcium infarction & septic embolization to other organs. oxalate crystals in the HPE specimen. © by author ESCMID Online Lecture Library PAS Chowdhary A, Randhawa H.S., Khanna G, Chakravati A, Naglot A, Roy P. Occurrence and Etiology of Fungal-Rhinosinusitis in a New Delhi Teaching Hospital. International Journal of Infectious Diseases 12/2008; 12. 2. PAS stain

© by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Mucormycosis (zygomycosis) Morphology & staining Clinical spectrum & host D/Ds tissue response • Nonpigmented • Mucorales cause Molds which produce • Broad (5- to 20-µm) angioinvasive disease in nonpigmented hyphae in • Ribbon-like immunosuppressed hosts, tissues • Pauciseptate hyphae showing wherein the hyphal elements 1. Aspergillus spp. right-angled branching will be found amidst necrosis, 2. Other hyaline (Mucorales). hemorrhage, & vascular septate molds like • The hyphae may vary in width, thrombosis. Fusarium or appear folded or crinkled, and • May present as rhino Scedosporium. be sparse or fragmented. cerebral, pulmonary or • H&E, GMS & PAS demonstrate cutaneous disease, any of only the cell wall with no structure ©which by may disseminate.author inside. • Important to demonstrate invasionESCMID in the vessel wall or inOnline Lecture Library the lumen. H&E

 Naturally pigmented molds whose hyphae and conidia contain melanin.  Cause skin & S/T infections preceded by trauma.  Three associated clinical entities: Eumycetoma, Chromoblastomycosis & Phaeohyphomycosis.  Other associated clinical syndromes - onychomycosis, keratitis, allergic disease, pneumonia, brain abscesses, and disseminated disease. © by author  Respiratory disease by inhalation of conidia.  Dematiaceous fungi most frequently associated with eosinophilia & AFRS/ABPAESCMID are Bipolaris Online & Curvularia Lecture (clinicopathological Library features similar to Aspergillosis). Pathology: • Biopsy specimens should be obtained from areas with pigment.

• HPE shows pigmented , thin, 2-6 μm, hyphae, some irregularly swollen (toruloid or moniliform) with prominent septations & constrictions & terminal or intercalated vesicular swellings.

• In cases where no pigmentation on H&E , FM staining for demonstration of melanin pigment.

- S. Agarwal, M.R. Capoor, V. Ramesh, Rajni, G. Khanna. First case of Acremonium kiliense mycetoma in a New Delhi resident: A brief review. Journal De Mycologie Médicale. 2011;ESCMID 1.1016 Online Lecture Library - Azad K, Khanna G, Capoor MR, Gupta S. Cladophialophora carrionii: an etiological agent of cutaneous chromoblastomycosis from a non-endemic area, North India. Mycoses. 2009 Dec 17. Fusarium species in subcutaneous tissue showing an irregular dilated hyphae

Eumycetoma • Grains are interwoven mycelia lined by intensely eosinophilic material (Sp. Hoeppli phenomenon). • Dematiaceous fungi produce black grains while Scedosporium, Acremonium produce white grains. • Eumycotic mycetoma shows septate hyphae, 2-6 μm in dm, staining positive with GMS & PAS. • Actinomycotic mycetoma shows delicate, branched, gram positive filaments sometimes showing beading & meas. <1μm..

Chromoblastomycosis Pigmented round structures (“copper penny lesions” / “sclerotic bodies” / “muriform cells” ) with internal septations in multiple planes. © by author Intense epidermal HP & HK & pyogranulomatous response. Phaeohyphomycosis • Minimal changes in the epidermis. ESCMID• Cyst Onlinewall comprised Lecture of dense collagen Library with granulomatous inflammation. • Center has geographic necrosis with FB giant cells & fungal elements (yeasts & septated hyphae). PAS stain PAS stain

GMS stain

 Hyaline fungi difficult to observe in the keratin layer using H&E.  GMS or PAS required for identification.  HPE findings: - HK with focal PK. - Spongiosis & neutrophilic micro abscesses in acute cases. - Varying degrees of PV lymphocytes & plasma cells with prominent papillary© by author dermal edema. PAS - SevereESCMID inﬂammation of Onlinehair follicles Lecture Library and shafts (neutrophilic – kerion; mononuclear- Majocchi’s granuloma ) Inadequacies of HPE Reported sensitivity of pathological studies is more than 80%, however, it has some inadequacies:

1.On special staining a) Yeasts can be confused with neurosecretory granules & melanin. b) Hyphae can be confused with collagen fibers & basement membrane material. 2. Some transversally cut hyphae can look like yeasts that may even appear to be budding.© by author 3. Histopathology usually cannot provide the fungal genus and species. 4.ESCMID Multiple infections Online may be difficult Lecture to interpret. Library Causes for Positive Cultures but Negative HPE

1. The fungus present in the cultures is a colonizer or a contaminant.

2. The tissue is sampled from two different areas, and different samples are sent to microbiology and pathology. © by author 3. The pathologic specimen has not been extensively studied. ESCMID Online Lecture Library Causes for Positive HPE but Negative Cultures

1. When the tissue in the microbiology laboratory is ground too aggressively and the fungal cells are destroyed.

2. When the fungus in the tissue is not viable.

3. When the tissue is sampled© by from author two different areas & different samples are sent for HPE & culture. ESCMID Online Lecture Library To Conclude….

 In developing countries - major role, despite limitations, as improved culture facilities & molecular profiling of fungi, though ideal, are not widely available for routine diagnostic use.

 Microbiologists, Pathologists, and Clinicians need to be aware of the pitfalls of morphological diagnosis, and the alternative tests that can be performed to© make by organism author-specific diagnoses. ESCMID Online Lecture Library References & Resources

1. Schwarz J. Hum Pathol. 1982;13:519-533. 2. Watts JC. Am J Clin Pathol. 1994;102:711-712. 3. Renshaw AA. Am J Clin Pathol. 1994;102:736-740.8. 4. Tarrand JJ et al. Am J Clin Pathol. 2003;119:854-858. 5. Weydert JA et al. Arch Pathol Lab Med. 2007;131:780-783. 6. Watts JC et al. Am J Clin Pathol. 1998;109:1-2. 7. Guarner J, Brandt ME. Clin Microbiol Rev. 2011;24:247-80. 8. Das A et al. Histopathology. 2009;54:854-9. 9. Challa S et al. Eur Arch Otorhinolaryngol. 2010;267:1239-45. 10. Sangoi AR et al. Am J Clin Pathol. 2009;131:364-75. 11. Lee S et al. Med Mycol. 2010;48:886-8. © by author ESCMID Online Lecture Library